Hypercalcemia associated with malignant tumor is a serious complicated symptom found in 5 to 20% of the whole patients suffering from malignant tumor and is considered to be a terminal symptom of malignant tumor since it certainly leads to death if it is left as it is. Control of hypercalcemia may greatly affect the prognosis and QOL (quality of life) of a patient; therefore, it will clinically play a significant role.
Generally, hypercalcemia in patients suffering from malignant tumor is roughly classified between HHM (humoral hypercalcemia of malignancy) based on tumor-producing humoral bone resorption factors and LOH (local osteolytic hypercalcemia) based on local action of tumor transferred or infiltrated to the bone. In HHM, it is believed that bone resorption or osteoclasis is promoted to increase the flow of calcium and produces hypercalcemia in cooperation with the reduced renal calcium-excreting ability (S. Wada and N. Nagata, Internal Medicine, 69, 644-648).
Hypercalcemia is considered to exhibit its symptoms when the concentration of calcium in the serum exceeds 12 mg/dl; as its symptoms, anorexia (inappetence), nausea and emesis (vomiting) are non-specifically observed at the early stage in patients suffering from malignant tumor. When hypercalcemia is worsened, the reduction of water-concentrating ability due to lesion of the renal distal tubules leads to hyperuresis (polyuria) and anorexia and nausea will be accompanied with dehydration due to insufficient uptake of water.
As humoral factors causing HHM among the hypercalcemia associated with malignant tumor, Moseley, J. M. et al. found parathyroid hormone related protein (hereinafter referred to as “PTHrP”) which are substances like parathyroid hormone (PTH): Proc. Natl. Acad. Sci. USA (1987) 84, 5048-5052.
Thereafter, a gene coding for PTHrP was isolated (Suva, L. J. et al., Science (1987) 237, 893) and it was elucidated from its analysis that there are three kinds of human PTHrPs having 139, 141 and 173 amino acids due to alternative splicing of the gene and that various fragments are present in the blood due to restricted degradation of PTHrP (1-139) having the whole structure: Baba, H., Clinical Calcium (1995) 5, 229-223. In PTHrP, 8 amino acids of the N-terminal 13 amino acids are identical with those in PTH and it is deduced that the amino acid site at position 14 to position 34 has a steric structure similar to PTH as well; thus, PTHrP and PTH bind to a common PTH/PTHrP receptor at least in the N-terminal region: Jueppner, H. et al., Science (1991) 254, 1024-1026; Abou-Samra, A-B. et al., Proc. Natl. Acad. Sci. USA (1992) 89, 2732-2736.
PTHrP is reported to be produced in a variety of tumoral tissues and it has been elucidated that not only in tumors, PTHrP is also produced in various normal tissues of from fetuses to adults, including skin, central nervous system, uterus, placenta, lactating mammary gland, thyroid gland, parathyroid gland, adrenal gland, liver, kidney and urinary bladder: Burtis, W. J., Clin. Chem. (1992) 38, 2171-2183; Stewart, A. F. & Broadus, A. E., J. Clin. Endocrinol. (1991) 71, 1410-1414. Further, PTHrP is considered to play an important role in the metabolic regulation of calcium which is maintained at a higher level in the fetal to newborn period than in the mother.
PTH/PTHrP receptors are known to be present mainly in the bone and kidney (C. Shigeno, Clinical Calcium (1995) 5, 355-359) and to activate plural intracellular signal transmission systems by binding of PTHrP to the receptors. One of them is adenylate cyclase and the other is phospholipase C. Activation of adenylate cyclase increases the concentration of intracellular cAMP to activate protein kinase A. Phospholipase C decomposes phosphatidylinositol 4,5-bisphosphonate to produce inositol 1,4,5-triphosphonate and diacylglycerol. G-protein is involved in these signal transmission systems: Coleman, D. T. et al., Biochemical mechanisms of parathyroid hormone action. In: “The parathyroids” (Bilezikian, J. P. et al.), Raven Press, New York (1994) page 239.
Through these signal transmission systems, PTHrP causes hypercalcemia, hypophosphatemia, decrease of renal phosphate-resorbing ability, increase of renal cAMP-excretion and the like which are observed in HHM.
Thus, it has been elucidated that PTHrP is closely related to hypercalcemia associated with malignant tumor. In the treatment of hypercalcemia associated with malignant tumor, calcitonin, steroid agents, indomethacin, inorganic phosphate salts, bisphosphonates and the like are used, as well as fluid replacement. However, these agents may show reduction of their effects upon consecutive use, some serious side-effects, or slow expression of their pharmacological effects; accordingly, use of agents or drugs which have higher therapeutic effects and less side-effects is highly expected.
On the other hand, as a new attempt to treat hypercalcemia associated with malignant tumor, Kukreja, S. C. et al. reported that when a neutralizing antiserum against PTHrP was administered to athymic mice in which human lung or larynx cancer cells had been transplanted to generate hypercalcemia, the blood calcium concentration and urinary cAMP level were reduced: J. Clin. Invest. (1988) 82, 1798-1802. Kanji Sato et al. reported that when an antibody against PTHrP (1-34) was administered to nude mice to which a PTHrP-producing human tumor was transplanted, the hypercalcemia was reduced and the viable time period of the mice was greatly prolonged: J. bone & Mine. Res. (1993) 8, 849-860. Further, Japanese Patent Application Laid Open Publication No. 4-228089 discloses mouse/human chimeric antibodies against human PTHrP (1-34).
Mouse monoclonal antibodies are highly immunogenic (sometimes also referred to as “antigenic”) in humans, which limits the medical therapeutic values of the mouse monoclonal antibodies in humans. For instance, a mouse antibody may be metabolized as a foreign matter when administered to a human; therefore, the half-life of the mouse antibody is relatively short in humans and its expected effects are not sufficiently exhibited. Further, human anti-mouse antibodies (HAMA) raised against the administered mouse antibody may cause immune responses which are inconvenient and dangerous to patients, such as serum diseases and other allergic reactions. Accordingly, mouse monoclonal antibodies can not frequently be administered to humans.
In order to solve these problems, methods for reducing the immunogenicity of non-human derived antibodies, for example, mouse-derived monoclonal antibodies have been developed. One of these methods is to make a chimeric antibody in which the variable-region (V region) is derived from a mouse monoclonal antibody and the constant region (C region) is derived from an appropriate human antibody.
Since the resulting chimeric antibody has the intact variable region of the original mouse antibody, it can be expected that the chimeric antibody may bind to an antigen with the same specificity as the original mouse antibody. Further, such a chimeric antibody has a substantially reduced proportion of an amino acid sequence derived from a non-human animal; therefore, it is anticipated to have a lower immunogenicity as compared with the original mouse antibody. Although the chimeric antibody binds to its antigen equivalently with the original mouse monoclonal antibody while showing a lower immunogenicity, some immune responses to the mouse variable region may still be possibly generated: LoBuglio, A. F. et al., Proc. Natl. Acad. Sci. USA, 86, 4220-4224, 1989.
A second method for reducing the immunogenicity of mouse antibodies is still more complicated but expected to further greatly reduce the potential immunogenicity of the mouse antibodies. In this method, only the complementarity determining regions (CDRs) of the variable region of a mouse antibody are grafted to a human variable region to create a “reshaped” human variable region. If required, a partial amino acid sequence of a framework region (FR) supporting the CDRs in a variable region of a mouse antibody may be grafted to a human variable region in order to make the structure of CDRs in the reshaped human variable region closer to that of the original mouse antibody.
Then, these humanized, reshaped human variable regions are combined with human constant regions. In the finally reshaped, humanized antibody, the portions derived from non-human amino acid sequences are only CDRs and a very small part of FR. The CDRs are composed of a hypervariable amino acid sequence and these do not show any species-specific sequences. Therefore, a humanized antibody comprising mouse CDRs will no longer have any stronger immunogenicity than a naturally occurring human antibody containing human CDRs.
With respect to humanized antibodies, further reference should be made to Riechmann, L. et al., Nature, 332, 323-327, 1988; Verhoeye, M. et al., Science, 239, 1534-1536, 1988; Kettleborough, C. A. et al., Protein Engng., 4, 773-783, 1991; Maeda, H. et al., Human Antibodies and Hybridoma, 2, 124-134, 1991; Gorman, S. D. et al., Proc. Natl. Acad. Sci. USA, 88, 4181-4185, 1991; Tempest, P. R. et al., Bio/Technology, 9, 266-271, 1991; Co, M. S. et al., Proc. Natl. Acad. Sci. USA, 88; 2869-2873, 1991; Carter, P. et al., Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148, 1149-1154 1992; and Sato, K. et al., Cancer Res., 53, 851-856, 1993.
Although humanized antibodies are expected to be useful for therapeutic purposes as previously mentioned, no humanized antibody against PTHrP has been known nor suggested in the aforementioned references. Further, there is no standardized means generally applicable to any antibodies in the process for preparing humanized antibodies; various means and methods are necessary to make a humanized antibody exhibiting a sufficient binding, neutralizing activity to a specific antigen: see, for example, Sato, K. et al., Cancer Res., 53, 851-856, 1993.